PROJECT SUMMARY Irritable bowel syndrome (IBS) is a functional gastrointestinal disorder which affects up to 20% of school- children worldwide; it exerts a tremendous social, emotion, and economic burden. Dietary interventions, particularly those removing fermentable carbohydrates (CHOs) from the diet, have demonstrated efficacy in both adults and children with IBS. Among fermentable CHOs, fructans are naturally occurring and abundant in the diet. After ingestion, fructans arrive in the colon essentially intact where they are fermented rapidly by gut microbiota. My preliminary data has found that a subset (~50%) of children with IBS is fructan sensitive (Fsen) - experience worsening abdominal pain, flatulence, and bloating when fed fructans. The exact mechanism behind fructan sensitivity in IBS is unknown but is thought to be related, in part, to fructan fermentation by the gut microbiome. The gut microbiome is composed of several organisms including bacteria, fungi, and archaea. There is a growing consensus that the gut microbiome contributes to the pathogenesis of IBS; however, studies to date have focused solely on the potential role of bacteria. Yet, in addition to bacteria, both fungi and archaea can ferment CHOs (such as fructans) and also regulate bacterial CHO fermentation. Preliminary data suggest that the gut fungal and archaeal composition differs between those children with IBS who do versus do not improve on a low fermentable CHO diet. Given: (a) A clinically significant proportion of children with IBS are fructan sensitive; (b) Both fungi and archaea can play a role in CHO (e.g., fructan) fermentation; and (c) Preliminary evidence of differential abundance of gut fungi and archaea relative to clinical response to a low CHO diet, the current R03 proposal aims to leverage my ongoing K23 study (currently focused on bacteria) to elucidate the potential role of fungi and archaea in dietary fructan-induced GI symptoms. I will take advantage of existing fecal samples from my ongoing childhood IBS randomized, double blind, crossover, placebo controlled fructan vs. maltodextrin (placebo) challenge study to perform specific fungal and archaeal metagenomic analyses. Before and after the dietary fructan vs. placebo challenge, the gut mycobiome (fungal) composition (Aim 1) and archaeal composition (Aim 2) will be compared between Fsen vs. fructan insensitive (Fins) subjects. We hypothesize that both gut fungal and archaeal composition will differ between Fsen and Fins groups. The proposed approach is innovative - it will be the first study in IBS to characterize the nonbacterial (fungi and archaea) microbiome and its relationship to symptom generation in IBS; importantly it will be done within the context of a rigorous dietary intervention trial. The research is significant because it is likely to help lead to the development of new personalized strategies (e.g. dietary or microbiome-directed therapies) to address food- induced symptoms in IBS.